Malignant melanoma (stage 3) is a fatal disease killing most patients within one year of diagnosis. The incidence of melanoma is rapidly increasing in the United States and is even higher in other countries, such as Australia. Effective treatments for patients suffering from melanoma are urgently needed.
Kidney cancer currently kills approximately 13,000 individuals in the United States each year. This form of cancer is frequently not detected until it is well advanced. The only form of treatment that significantly affects a patient's prognosis is surgical resection of the affected organ. Unfortunately, because this type of cancer is highly metastatic, complete removal of all the metastasis is difficult, if not impossible.
Colon cancer is one of the most prevalent forms of cancer and currently kills approximately 140,000 individuals in the United States each year. Although there have been a large number of traditional chemotherapeutic drugs developed to treat this disease, long term survival (defined as the percentage of patients surviving five years or more) has not appreciably changed in the last four decades. Furthermore, all of the traditional chemotherapeutic drugs developed are highly toxic, have deleterious and often fatal side effects, and are expensive. A curative, non-toxic treatment for this disease is urgently needed.
A hallmark of melanomas, kidney and colon tumors is that these tumors quickly develop resistance to traditional chemotherapies. Even though patients may initially respond to chemotherapeutic treatment, drug-resistant tumors quickly arise and often kill the patient. An alternative way to treat these tumors would be to identify an “Achilles Heel” in the tumors and to develop therapies that would selectively treat that target. One such potential target has been identified. Specifically, it has been noted that all three of these types of tumors require extensive vascularization of each of the metastacies in order for the cancers to grow. Therefore, one would predict that a therapeutic agent which would inhibit the vascularization of these tumors may provide a unique means of treating these tumors.
Tumor necrosis factor (TNF) is a cytokine originally named for its ability to kill tumors. There are at least two different mechanisms by which TNF is believed to kill tumors. First is by a direct effect on the tumor itself. Second, TNF can selectively disrupt the vascularization of tumors, thus depriving the tumor of nutrients and oxygen and in so doing killing the tumor indirectly. This latter mechanism of killing was described in the first scientific publication describing TNF. Carswell and Old reported that the METH A tumor cells were completely resistant to TNF in vitro. J. Proc. Natl. Acad. Sci USA, 72:3666-3670 (1975). However, METH A tumors in mice were extremely sensitive to killing by TNF in vivo. It was later shown that TNF selectively disrupted the vascularization of these METH A tumors. Subsequently it was later shown that a factor (EMAP 2) is released by some tumors that renders the tumor vasculature susceptible to TNF killing. Thus, TNF can kill some tumors (such as METH A sarcomas) not by directly killing the tumor cells, but rather by killing the tumors' vasculature that provides the tumor with blood, oxygen and other nutrients necessary to live and grow.
Early clinical trials attempted to utilize TNF as a direct tumoricidal agent. This coupled with the fact that because TNF has a very short circulating half life (less than 20 minutes) in the circulation, extremely high doses of TNF were used which induced “shock”-like symptoms characterized by a precipitous drop in blood pressure and often death of the patient.
An alternative method of using TNF would be to formulate it so that it remains in the circulation longer thus giving it more time to react with (and thus destroy) the vasculature of the tumors. Several other therapeutic proteins which had very short circulating half lives have been formulated with polyethylene glycol (PEG) so that they circulate longer and remain in the vasculature. These proteins include asparaginase, adenosine deaminase, and super oxide dismutase. See, for example, Harras, J. M., in “Polyethylene Glycol Chemistry: Biotechnical and Biochemical Applications,” Plenum Press (1992).
Relevant to the invention described here, a group of investigators in Japan (Tsutsumi et. al.) have described that TNF could be formulated with certain PEG and that the resulting material had substantially increased circulating half-life and greater anti-tumor activity. See, Tsutsumi, Y., et al., Jap. J. Cancer Res., 85:9-12 (1994); Tsutsumi, Y., et al., Jap. J. Cancer Res., 85:1185-1188 (1994); Tsutsumi, Y., et al., Jap. J. Cancer Res., 87:1078-1085 (1997). However these investigators used only PEG with a molecular weight of 5000 (PEG5000) attached to the primary amines on TNF with a succinimidyl succinate linker and failed to determine not only the optimal method of attaching PEG to TNF but also the optimal attachment sites on the molecule.